CN106967169B - Extraction method of fish collagen - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
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Abstract
The invention relates to a method for extracting fish collagen, belonging to the technical field of natural protein extraction. The invention adopts a fish collagen extraction process without using organic solvent to realize the removal and separation of fat, and the technical concept is mainly as follows: firstly, grinding and homogenizing under the action of ultrasonic wave to fully break the tissue cells of the fish skin, so that fat and protein can be removed more in the subsequent alkali treatment and enzyme treatment processes to obtain a crude extract with low fat content, and then, passing the crude extract solution through a oleophylic modified resin bed to realize the purpose of adsorbing the low-content lipid. The prepared fish collagen has the advantages of high purity, low fat content and good water solubility.
Description
Technical Field
The invention relates to a method for extracting fish collagen, belonging to the technical field of natural protein extraction.
Background
Collagen (collagen) is a white, opaque, unbranched fibrous protein that plays a role as a binding tissue in animal cells. Collagen is rich in 18 kinds of amino acids other than tryptophan and cysteine, and 7 kinds of amino acids are necessary for maintaining the growth of human body. The collagen contains 30% of glycine and 25% of proline and hydroxyproline, and has the highest content of various proteins, and the content of alanine and glutamic acid is also high. Meanwhile, hydroxyproline and pyroglutamic acid, which are rare in general proteins, and hydroxylysine, which hardly exists in other proteins, are also contained. Therefore, the collagen is rich in nutrition. The fish collagen is mainly extracted from fresh skin and fish scales of tilapia, cod and cod through a biological enzyme directional shearing technology, has a molecular weight of 2000 Da-5000 Da, has high digestion and absorption, excellent thermal stability and excellent biocompatibility, and can well play the functions of permeation, moisture preservation, repair and the like.
The fish collagen mainly contains type I and type V collagen, is distributed on skin, bone, scale, etc. of fish, and is spiral fibrous protein formed by twisting 3 peptide chains. The main amino acid compositions of the polypeptide are glycine, proline, hydroxyproline, alanine, hydroxylysine and the like, the amino acid compositions are obviously different from other proteins, the content of the glycine is very high and almost accounts for one third of the total amino acid residues, namely, 1 glycine is arranged at every 2 other amino acid residues (X, Y), so the peptide chain can be expressed by (Gly-X-Y) n; it contains hydroxyproline and hydroxylysine residues that are rare among other proteins. Due to the particularity of marine ecological environment (such as high pressure, low temperature and the like), the fish collagen has certain difference in amino acid composition and amino acid sequence compared with the collagen of terrestrial animals, so that the fish collagen has unique physiological function and physicochemical property. The fish collagen is easy to dissolve in neutral salt solution or dilute acid, and is easy to be prepared into soluble collagen solution, and because of the characteristic of low antigenicity of the fish collagen, the fish collagen is more popular with people in the fields of medical treatment and food industry.
Taking 2009 as an example, the total yield of fish is 2990 ten thousand t, and the number of waste fish bones is as high as 1000 ten thousand t, which indicates that the byproduct resources of the fish product are very rich. If the waste fishbone is deeply processed to develop a high-tech product and the depth of the fishery product combination scheme is excavated, the development level of fishery industry in China can be greatly improved, and further more market share is occupied, and the method has a very wide development prospect. Therefore, the collagen extracted from the fish by-products not only can fully utilize resources and improve the added value of fish processing, but also can reduce environmental pollution, and has important significance for promoting the development of the fish processing industry.
To date, researchers at home and abroad have reported many methods for extracting collagen. Generally, fish collagen extraction methods can be classified into five types according to the difference of extraction media: hot water method, acid method, alkaline method, salt method, enzymatic method, and the like. In either method, the basic principle is to separate collagen from other proteins or matrices by changing the environment in which the proteins are located based on the properties of the collagen. In many cases, a single extraction method does not reach an intended target, so in actual practice, a plurality of extraction methods are generally combined with each other.
Defatting and removing impurity protein before extracting and separating collagen, and mixing with lipidForming an opacified collagen solution, making lipid removal difficult; the mixing of foreign proteins in the collagen will affect the efficiency of subsequent separation and extraction, and thus the purity of the collagen product. Degreasing is very necessary in the extraction and separation processes of fish collagen, and the existing fish collagen degreasing method comprises the following steps: (1) the organic solvent extraction method is the most common method for degreasing the fish collagen, the solvent can be recovered, the cost is saved, the environmental pollution is reduced, and the currently applied solvents are methanol, normal hexane, diethyl ether, ethanol, isopropanol, acetone and the like. For example, the purity of collagen is improved by removing fat contained in fish skin with ethanol, acetone and n-hexane. However, the organic solvent has toxicity and is harmful to human bodies if not removed cleanly. (2) Alkaline solution degreasing method, alkaline solution can be saponified with fat, remove fat effectively. For example, the fish scale is taken as a raw material, sodium bicarbonate solution is added for degreasing, and low-fat fish scale collagen is obtained. However, the alkali concentration must be strictly controlled so as not to destroy the triple helix structure of fish collagen. (3) CO 22Supercritical processor degreasing, CO2Supercritical processing can avoid these disadvantages. Extracting flea white with hydrolytic proteinase, and treating the hydrolytic solution with CO2After supercritical degreasing and fish skin treatment, the oil content of the fish skin is reduced to a great extent, meanwhile, the effects of deodorization, decoloration and deodorization are achieved, the purity of collagen is improved, and the triple-helix structure of the collagen can be well maintained. (4) The lipase method mainly utilizes the hydrolysis of fat molecules by lipase, and the hydrolysis is easy to emulsify and remove.
Disclosure of Invention
The invention aims to solve the problems of long operation time and serious pollution caused by the need of using more organic solvents in the separation process of lipid substances and protein in the extraction process of fish collagen; the invention adopts a fish collagen extraction process without using organic solvent to realize the removal and separation of fat, and the technical concept is mainly as follows: firstly, grinding and homogenizing under the action of ultrasonic wave to fully break the tissue cells of the fish skin, so that fat and protein can be removed more in the subsequent alkali treatment and enzyme treatment processes to obtain a crude extract with low fat content, and then, passing the crude extract solution through a oleophylic modified resin bed to realize the purpose of adsorbing the low-content lipid.
The technical scheme is as follows:
a method for extracting fish collagen comprises the following steps:
step 1, tissue disruption: after thawing the skin, adding water to a weight ratio of skin to water of 10: 0.2-1, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with an alkali solution, uniformly stirring, soaking, and centrifuging to remove the supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with an acid solution, uniformly stirring, soaking, centrifuging, and removing a supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, adding protease accounting for 1-5% of the weight of the third precipitate for enzymolysis, heating to inactivate enzyme after the reaction is finished, and centrifuging to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid;
and 7, refining treatment: adding solid NaCl into the degreasing solution to enable the concentration of the NaCl to be 0.5-1.5 mol/L, and performing refrigerated centrifugation to obtain a salted-out collagen precipitate; re-dissolving the collagen precipitate with 0.5-1.5 mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain the refined fish collagen.
In the step 1, the fish skin is selected from carp, crucian carp, black carp, grass carp, silver carp, bighead carp or tilapia.
In the step 2, the mass concentration of the NaCl solution is 4-10%, and the weight ratio of the slurry to the NaCl solution is 1: 5-10, and the soaking time is 10-30 h.
In the step 3, the alkali solution is selected from NaOH solution, KOH solvent and NaHCO3Solutions or K2CO3A solution; the mass concentration of the alkali solution is 2-5%, and the weight ratio of the first precipitate to the alkali solution is 1: 7-12 and soaking time is 20-35 h.
In the step 4, the acid solvent is selected from citric acid, acetic acid or oxalic acid, the mass concentration of the acid solvent is 5-12%, and the weight ratio of the second precipitate to the acid solution is 1: 4-8, and the soaking time is 5-10 h.
In the step 5, the weight ratio of the third precipitate to the water is 1: 15-35, the protease refers to papain, bromelain, alkaline protease or trypsin, the enzymolysis temperature is 35-45 ℃, the enzymolysis time is 4-10 hours, and the pH of the reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process; the enzyme deactivation is to treat the mixture at 90-95 ℃ for 5-15 min.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 10-15 parts by weight of microcrystalline cellulose, 150-200 parts by weight of water and 0.5-1 part by weight of a dispersing agent into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 5-10 parts of butyl methacrylate, 2-4 parts of Glycidyl Methacrylate (GMA), 0.3-0.6 part of cross-linking agent, 1.5-3 parts of pore-forming agent and 15-20 parts of water, heating to 75-85 ℃ for reaction for 4-8 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, and then carrying out vacuum drying and crushing to obtain the degreasing resin.
The dispersant is polyvinyl alcohol; the cross-linking agent is ethylenediamine, butanediamine, carbodiimide, trimethylolmelamine, dimethylol urea or aziridine; the pore-foaming agent is ethyl acetate.
In the step 6, the volume of the resin is 1/40-1/20 of the volume of the crude extract, the flow rate of the crude extract is 3-5 BV/h, and the temperature in the adsorption process is 20-35 ℃.
In the resin, microcrystalline cellulose is mainly used as a main component for absorbing the fat, the surface of the resin is subjected to macroporous treatment by a pore-forming agent, butyl methacrylate monomer is used for grafting modification to improve the fat absorption effect, glycidyl methacrylate monomer is used for modification to improve the repulsion force to the protein, and the protein yield is improved.
Advantageous effects
The invention adopts a fish collagen extraction process without using organic solvent to realize the removal and separation of fat, and the technical concept is mainly as follows: firstly, grinding and homogenizing under the action of ultrasonic wave to fully break the tissue cells of the fish skin, so that fat and protein can be removed more in the subsequent alkali treatment and enzyme treatment processes to obtain a crude extract with low fat content, and then, passing the crude extract solution through a oleophylic modified resin bed to realize the purpose of adsorbing the low-content lipid. The prepared fish collagen has the advantages of high purity, low fat content and good water solubility.
Drawings
FIG. 1 is a graph of the solubility of fish collagen at different pH conditions.
Detailed Description
Example 1
Step 1, tissue disruption: after the crucian skin is unfrozen, adding water, wherein the weight ratio of the crucian skin to the water is 10: 0.2, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with a 2wt% NaOH solution, wherein the weight ratio of the first precipitate to the alkali solution is 1: 7, uniformly stirring, soaking for 20 hours, and centrifuging to remove the supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with a 5wt% citric acid solution, wherein the weight ratio of the second precipitate to the acid solution is 1: 4, uniformly stirring and soaking for 5 hours, and then centrifuging to remove supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, wherein the weight ratio of the third precipitate to the water is 1: 15, adding papain with the weight of 1% of that of the third precipitate for enzymolysis, wherein the enzymolysis temperature is 35 ℃, the enzymolysis time is 4 hours, the pH of a reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process, treating at 90 ℃ for 5min for enzyme deactivation after the reaction is finished, and centrifuging to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid, wherein the volume of the resin is 1/40 of the volume of the crude extract, the flow rate of the crude extract is 3BV/h, and the temperature in the adsorption process is 20 ℃;
and 7, refining treatment: adding solid NaCl into the degreasing solution to make the concentration of NaCl be 0.5mol/L, and freezing and centrifuging to obtain salted-out collagen precipitate; re-dissolving the collagen precipitate with 0.5mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 10 parts of microcrystalline cellulose, 150 parts of water and 0.5 part of dispersant polyvinyl alcohol into a reaction kettle, heating and uniformly stirring; and under the nitrogen atmosphere, adding 5 parts of butyl methacrylate, 2 parts of Glycidyl Methacrylate (GMA), 0.3 part of cross-linking agent ethylenediamine, 1.5 parts of pore-forming agent ethyl acetate and 15 parts of water, heating to 75 ℃ to react for 4 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, drying in vacuum, and crushing to obtain the degreasing resin.
Example 2
Step 1, tissue disruption: after the crucian skin is unfrozen, adding water, wherein the weight ratio of the crucian skin to the water is 10: 1, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with a 5wt% NaOH solution, wherein the weight ratio of the first precipitate to the alkali solution is 1: 12, uniformly stirring, soaking for 35 hours, and centrifuging to remove supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with a 12wt% citric acid solution, wherein the weight ratio of the second precipitate to the acid solution is 1: 8, uniformly stirring, soaking for 10 hours, centrifuging, and removing a supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, wherein the weight ratio of the third precipitate to the water is 1: 35, adding papain with the weight of 5% of that of the third precipitate for enzymolysis, wherein the enzymolysis temperature is 45 ℃, the enzymolysis time is 10 hours, the pH of a reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process, treating at 95 ℃ for 15min for enzyme deactivation after the reaction is finished, and centrifuging to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid, wherein the volume of the resin is 1/20 of the volume of the crude extract, the flow rate of the crude extract is 5BV/h, and the temperature in the adsorption process is 35 ℃;
and 7, refining treatment: adding solid NaCl into the degreasing solution to make the concentration of NaCl be 1.5mol/L, and freezing and centrifuging to obtain salted-out collagen precipitate; re-dissolving the collagen precipitate with 1.5mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 15 parts of microcrystalline cellulose, 200 parts of water and 1 part of dispersant polyvinyl alcohol into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 10 parts of butyl methacrylate, 4 parts of Glycidyl Methacrylate (GMA), 0.6 part of cross-linking agent ethylenediamine, 3 parts of pore-forming agent ethyl acetate and 20 parts of water, heating to 85 ℃ to react for 8 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, drying in vacuum, and crushing to obtain the degreasing resin.
Example 3
Step 1, tissue disruption: after the crucian skin is unfrozen, adding water, wherein the weight ratio of the crucian skin to the water is 10: 0.6, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with a 4wt% NaOH solution, wherein the weight ratio of the first precipitate to the alkali solution is 1: 11, uniformly stirring, soaking for 26 hours, and centrifuging to remove supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with 5-12 wt% of citric acid solution, wherein the weight ratio of the second precipitate to the acid solution is 1: 6, uniformly stirring and soaking for 8 hours, and then centrifuging to remove supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, wherein the weight ratio of the third precipitate to the water is 1: 20, adding papain with the weight of 3% of that of the third precipitate for enzymolysis, wherein the enzymolysis temperature is 40 ℃, the enzymolysis time is 7 hours, the pH of a reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process, treating the reaction solution at 92 ℃ for 10min for enzyme deactivation after the reaction is finished, and centrifuging the reaction solution to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid, wherein the volume of the resin is 1/30 of the volume of the crude extract, the flow rate of the crude extract is 4BV/h, and the temperature in the adsorption process is 25 ℃;
and 7, refining treatment: adding solid NaCl into the degreasing solution to enable the concentration of the NaCl to be 1mol/L, and freezing and centrifuging to obtain salted-out collagen precipitate; re-dissolving the collagen precipitate with 1mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 12 parts of microcrystalline cellulose, 170 parts of water and 0.7 part of dispersant polyvinyl alcohol into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 6 parts of butyl methacrylate, 3 parts of Glycidyl Methacrylate (GMA), 0.5 part of cross-linking agent ethylenediamine, 2 parts of pore-forming agent ethyl acetate and 17 parts of water, heating to 80 ℃ to react for 7 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, drying in vacuum, and crushing to obtain the degreasing resin.
Comparative example 1
The difference from example 3 is that: the monomer butyl methacrylate is not added in the preparation of resin degreasing, and the weight of the monomer butyl methacrylate is replaced by glycidyl methacrylate.
Step 1, tissue disruption: after the crucian skin is unfrozen, adding water, wherein the weight ratio of the crucian skin to the water is 10: 0.6, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with a 4wt% NaOH solution, wherein the weight ratio of the first precipitate to the alkali solution is 1: 11, uniformly stirring, soaking for 26 hours, and centrifuging to remove supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with 5-12 wt% of citric acid solution, wherein the weight ratio of the second precipitate to the acid solution is 1: 6, uniformly stirring and soaking for 8 hours, and then centrifuging to remove supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, wherein the weight ratio of the third precipitate to the water is 1: 20, adding papain with the weight of 3% of that of the third precipitate for enzymolysis, wherein the enzymolysis temperature is 40 ℃, the enzymolysis time is 7 hours, the pH of a reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process, treating the reaction solution at 92 ℃ for 10min for enzyme deactivation after the reaction is finished, and centrifuging the reaction solution to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid, wherein the volume of the resin is 1/30 of the volume of the crude extract, the flow rate of the crude extract is 4BV/h, and the temperature in the adsorption process is 25 ℃;
and 7, refining treatment: adding solid NaCl into the degreasing solution to enable the concentration of the NaCl to be 1mol/L, and freezing and centrifuging to obtain salted-out collagen precipitate; re-dissolving the collagen precipitate with 1mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 12 parts of microcrystalline cellulose, 170 parts of water and 0.7 part of dispersant polyvinyl alcohol into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 6 parts of Glycidyl Methacrylate (GMA), 0.5 part of cross-linking agent ethylenediamine, 2 parts of pore-forming agent ethyl acetate and 17 parts of water, heating to 80 ℃ to react for 7 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, drying in vacuum, and crushing to obtain the degreasing resin.
Comparative example 2
The difference from example 3 is that: the monomer glycidyl methacrylate is not added in the preparation of resin degreasing, and the weight of the monomer glycidyl methacrylate is replaced by butyl methacrylate.
Step 1, tissue disruption: after the crucian skin is unfrozen, adding water, wherein the weight ratio of the crucian skin to the water is 10: 0.6, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with a 4wt% NaOH solution, wherein the weight ratio of the first precipitate to the alkali solution is 1: 11, uniformly stirring, soaking for 26 hours, and centrifuging to remove supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with 5-12 wt% of citric acid solution, wherein the weight ratio of the second precipitate to the acid solution is 1: 6, uniformly stirring and soaking for 8 hours, and then centrifuging to remove supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, wherein the weight ratio of the third precipitate to the water is 1: 20, adding papain with the weight of 3% of that of the third precipitate for enzymolysis, wherein the enzymolysis temperature is 40 ℃, the enzymolysis time is 7 hours, the pH of a reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process, treating the reaction solution at 92 ℃ for 10min for enzyme deactivation after the reaction is finished, and centrifuging the reaction solution to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid, wherein the volume of the resin is 1/30 of the volume of the crude extract, the flow rate of the crude extract is 4BV/h, and the temperature in the adsorption process is 25 ℃;
and 7, refining treatment: adding solid NaCl into the degreasing solution to enable the concentration of the NaCl to be 1mol/L, and freezing and centrifuging to obtain salted-out collagen precipitate; re-dissolving the collagen precipitate with 1mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 12 parts of microcrystalline cellulose, 170 parts of water and 0.7 part of dispersant polyvinyl alcohol into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 9 parts of butyl methacrylate, 0.5 part of cross-linking agent ethylenediamine, 2 parts of pore-forming agent ethyl acetate and 17 parts of water, heating to 80 ℃ to react for 7 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, drying in vacuum, and crushing to obtain the degreasing resin.
Comparative example 3
The difference from example 3 is that: no sonication was added in step 1.
Step 1, tissue disruption: after the crucian skin is unfrozen, adding water, wherein the weight ratio of the crucian skin to the water is 10: 0.6, crushing and grinding the mixture to obtain slurry;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with a 4wt% NaOH solution, wherein the weight ratio of the first precipitate to the alkali solution is 1: 11, uniformly stirring, soaking for 26 hours, and centrifuging to remove supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with 5-12 wt% of citric acid solution, wherein the weight ratio of the second precipitate to the acid solution is 1: 6, uniformly stirring and soaking for 8 hours, and then centrifuging to remove supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, wherein the weight ratio of the third precipitate to the water is 1: 20, adding papain with the weight of 3% of that of the third precipitate for enzymolysis, wherein the enzymolysis temperature is 40 ℃, the enzymolysis time is 7 hours, the pH of a reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process, treating the reaction solution at 92 ℃ for 10min for enzyme deactivation after the reaction is finished, and centrifuging the reaction solution to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid, wherein the volume of the resin is 1/30 of the volume of the crude extract, the flow rate of the crude extract is 4BV/h, and the temperature in the adsorption process is 25 ℃;
and 7, refining treatment: adding solid NaCl into the degreasing solution to enable the concentration of the NaCl to be 1mol/L, and freezing and centrifuging to obtain salted-out collagen precipitate; re-dissolving the collagen precipitate with 1mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen.
In the step 6, the preparation method of the degreasing resin comprises the following steps: adding 12 parts of microcrystalline cellulose, 170 parts of water and 0.7 part of dispersant polyvinyl alcohol into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 6 parts of butyl methacrylate, 3 parts of Glycidyl Methacrylate (GMA), 0.5 part of cross-linking agent ethylenediamine, 2 parts of pore-forming agent ethyl acetate and 17 parts of water, heating to 80 ℃ to react for 7 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, drying in vacuum, and crushing to obtain the degreasing resin.
Determination of yield
Respectively measuring the protein content in the crude extract and the refined fish collagen obtained in the step 5 by adopting a Kjeldahl method, converting the protein content into the protein mass, calculating the resin adsorption yield, and calculating by adopting the following formula:
the resin adsorption yield = protein content in the refined fish collagen × weight of the refined fish collagen/(weight of protein content in crude extract) × 100%.
As can be seen from the above table, when the resin is used for adsorbing lipid impurities, more protein can be ensured to permeate the resin, so that the ingestion rate of the fish collagen is higher; example 3 compared with comparative example 2, the repulsion force to protein can be improved by adding glycidyl methacrylate in the preparation of resin, so that the protein is prevented from being adsorbed by the resin, and the yield is improved.
Ultraviolet spectral analysis
Ultraviolet absorption peaks of the extracted collagen are analyzed by adopting an ultraviolet spectrum. The collagen sample was dissolved in 0.5M acetic acid solution to prepare a collagen solution with a concentration of 1 mg/mL. The absorbance was measured at room temperature with an ultraviolet-visible spectrophotometer. The scanning wavelength range is 200-400 nm, and the wavelength precision is 1 nm.
The maximum absorption wavelengths of the fish collagen samples prepared in the above examples and comparative examples are as follows:
as can be seen from the above table, the maximum absorption wavelength peaks are all at 230nm, consistent with the characteristic UV absorption of type I collagen. Among them, the ultraviolet light absorption curve in example 13 has no hetero peak, showing that the fish collagen has a high purity.
Analysis of amino acid composition
The amino acid composition of the collagen sample was determined by high performance liquid chromatography. The measurement conditions were as follows: sopium amino acid Analysis chromatographic column, gradient elution, phase A of 0.2mol/L sodium citrate water solution with pH =3.00, phase B of 0.2mol/L sodium borate water solution with pH =9.80, eluent flow rate of 0.4mL/min, column temperature of 65 ℃.
Hydroxylation rate% = number of hydroxyproline residues × 100/(number of proline residues + number of hydroxyproline residues)
Solubility determination
The Nitrogen Solubility Index (NSI) can be used to determine the solubility of protein hydrolysates. Taking the supernatant of 0.01g/mL of the sea bass collagen peptide solution. And (3) measuring the nitrogen content in the supernatant and the collagen peptide sample by using a full-automatic Kjeldahl azotometer to obtain the water-soluble protein content of the collagen peptide sample under each pH value. And drawing an NSI-pH curve as an index of the solubility of the collagen peptides with different components under different pH values.
The nitrogen solubility index NSI is calculated as follows:
NSI（%）=A/B×100%
wherein A is the protein content (g/g) in the supernatant; b is the total protein content (g/g) in the sample.
The solubility under different pH conditions is shown in FIG. 1, and it can be seen from the figure that the fish collagen prepared in examples 1-3 has better solubility, which indicates that the content of impurities is low.
The solubility data are shown in the following table:
as can be seen from the table, in example 3, compared with comparative example 1, the microcrystalline cellulose is not modified by butyl methacrylate monomer, so that the absorption of fat is low, more fat exists in the extracted protein, and the solubility is poor. Compared with the comparative example 3, in the example 3, the treatment of the tissue disruption process by using ultrasound is not adopted, so that fat cannot be removed in the subsequent acid-base enzymolysis treatment process, the subsequent resin degreasing process has a large load, and the fat cannot be completely removed, so that the solubility is poor.
Fat content determination
Measuring the fat content in the fish collagen by adopting a chloroform-methanol extraction method, if the following steps are adopted:
as can be seen from the table, the fish collagen obtained by the process has lower fat content, and compared with the comparative example 1, the example 3 has the advantage that the removal rate of fat can be effectively improved by modifying microcrystalline cellulose by butyl methacrylate monomer; likewise, the use of ultrasonic pretreatment can reduce the burden of the subsequent resin degreasing process.
Claims (8)
1. A method for extracting fish collagen is characterized by comprising the following steps:
step 1, tissue disruption: after thawing the skin, adding water to a weight ratio of skin to water of 10: 0.2-1, crushing and grinding the mixture, and adding ultrasonic action while crushing and grinding to obtain slurry;
step 2, removing salt-soluble impurities: mixing the slurry with a NaCl solution, uniformly stirring, soaking, centrifuging, and removing a supernatant to obtain a first precipitate;
and 3, removing lipid and alkali-soluble impurities: mixing the first precipitate with an alkali solution, uniformly stirring, soaking, and centrifuging to remove the supernatant to obtain a second precipitate;
and 4, removing acid-soluble impurities: mixing the second precipitate with an acid solution, uniformly stirring, soaking, centrifuging, and removing a supernatant to obtain a third precipitate;
and 5, enzymolysis treatment: mixing the third precipitate with water, adding protease accounting for 1-5% of the weight of the third precipitate for enzymolysis, heating to inactivate enzyme after the reaction is finished, and centrifuging to obtain a supernatant to obtain a crude extract;
and 6, resin degreasing treatment: feeding the crude extract into a degreasing resin bed layer for adsorption treatment to obtain degreasing liquid;
and 7, refining treatment: adding solid NaCl into the degreasing solution to enable the concentration of the NaCl to be 0.5-1.5 mol/L, and performing refrigerated centrifugation to obtain a salted-out collagen precipitate; re-dissolving the collagen precipitate with 0.5-1.5 mol/L acetic acid, bagging, dialyzing to remove salt ions, and freeze-drying to obtain refined fish collagen;
in the step 6, the preparation method of the degreasing resin comprises the following steps: adding 10-15 parts by weight of microcrystalline cellulose, 150-200 parts by weight of water and 0.5-1 part by weight of a dispersing agent into a reaction kettle, heating and uniformly stirring; and then under the nitrogen atmosphere, adding 5-10 parts of butyl methacrylate, 2-4 parts of Glycidyl Methacrylate (GMA), 0.3-0.6 part of cross-linking agent, 1.5-3 parts of pore-forming agent and 15-20 parts of water, heating to 75-85 ℃ for reaction for 4-8 hours, after the reaction is finished, washing the solid with ethanol and water in sequence, and then carrying out vacuum drying and crushing to obtain the degreasing resin.
2. The method for extracting fish collagen according to claim 1, wherein in the step 1, the fish skin is selected from carp, crucian carp, black carp, grass carp, silver carp, bighead carp and tilapia.
3. The extraction method of fish collagen according to claim 1, wherein in the step 2, the mass concentration of the NaCl solution is 4-10%, and the weight ratio of the slurry to the NaCl solution is 1: 5-10, and the soaking time is 10-30 h.
4. The method for extracting fish collagen according to claim 1, wherein in the step 3, the alkali solution is selected from NaOH solution, KOH solution, NaHCO solution3Solutions or K2CO3A solution; the mass concentration of the alkali solution is 2-5%, and the weight ratio of the first precipitate to the alkali solution is 1: 7-12 and soaking time is 20-35 h.
5. The method for extracting fish collagen according to claim 1, wherein in the step 4, the acid solvent is selected from citric acid, acetic acid or oxalic acid, the mass concentration of the acid solvent is 5-12%, and the weight ratio of the second precipitate to the acid solution is 1: 4-8, and the soaking time is 5-10 h.
6. The method for extracting fish collagen according to claim 1, wherein in the step 5, the weight ratio of the third precipitate to water is 1: 15-35, the protease refers to papain, bromelain, alkaline protease or trypsin, the enzymolysis temperature is 35-45 ℃, the enzymolysis time is 4-10 hours, and the pH of the reaction solution is controlled to be 4-5 by HCl or NaOH in the enzymolysis process; the enzyme deactivation is to treat the mixture at 90-95 ℃ for 5-15 min.
7. The method for extracting fish collagen according to claim 1, wherein the dispersant is polyvinyl alcohol; the cross-linking agent is ethylenediamine, butanediamine, carbodiimide, trimethylolmelamine or dimethylol urea or aziridine; the pore-foaming agent is ethyl acetate.
8. The extraction method of fish collagen according to claim 1, wherein in the step 6, the volume of the resin is 1/40-1/20 of the volume of the crude extract, the flow rate of the crude extract is 3-5 BV/h, and the temperature of the adsorption process is 20-35 ℃.
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| CN107653288A (en) * | 2017-10-13 | 2018-02-02 | 金华市川璞农业科技有限公司 | With the purposes of catfish fish guts protease extraction collagen |
| CN107929091A (en) * | 2017-12-29 | 2018-04-20 | 大美生物科技有限公司 | A kind of extracting method of high activity collagen and its application |
| CN108486047B (en) * | 2018-02-13 | 2020-11-06 | 大美生物科技有限公司 | Medical dressing of stem cell extract and preparation method thereof |
| CN108740697A (en) * | 2018-05-24 | 2018-11-06 | 广西山水牛畜牧业有限责任公司 | A kind of processing method of ox-hide |
| CN109627325A (en) * | 2019-01-04 | 2019-04-16 | 广东海洋大学 | A kind of preparation method and applications of gradient collagen and collagen polypeptide molecule |
| CN113150612B (en) * | 2021-04-08 | 2023-03-21 | 西南大学 | Edible gelatin protein nano ink, preparation method and application thereof |
| CN114533856A (en) * | 2022-02-24 | 2022-05-27 | 浙江天妍生物科技有限公司 | Bioactive material injection for improving skin quality and preparation method and product thereof |
| CN114933648B (en) * | 2022-06-22 | 2023-09-08 | 山东恒鑫生物科技有限公司 | Method for producing collagen dipeptide from fish scales |
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| CN102703555A (en) * | 2012-06-08 | 2012-10-03 | 集美大学 | Extraction and preparation method for micromolecule fishskin collagen peptide |
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2017
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| CN102952837A (en) * | 2011-08-19 | 2013-03-06 | 上海华睿生物科技有限公司 | Medical fish collagen polypeptide raw material and its preparation technology |
| CN102703555A (en) * | 2012-06-08 | 2012-10-03 | 集美大学 | Extraction and preparation method for micromolecule fishskin collagen peptide |
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| Title |
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| Extraction and Characterization of Collagen from the Skin of Arothron stellatus Fish—A Novel Source of Collagen for Tissue Engineering;Ramanathan Giriprasath等;《Biomaterials and Tissue Engineering》;20141231;第4卷(第3期);1-7 * |
| 大孔树脂对罗非鱼皮胶原蛋白抗氧化肽脱盐作用的研究;夏光华等;《现代食品科技》;20131231;第29卷(第5期);1052-1056 * |
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